Design, Synthesis and Biological Evaluation of Novel Pleuromutilin Derivatives Containing 6-Chloro-1-R-1H-pyrazolo[3,4-d]pyrimidine-4-amino Side Chain

Two series of pleuromutilin derivatives were designed and synthesized as inhibitors against Staphylococcus aureus (S. aureus). 6-chloro-4-amino-1-R-1H-pyrazolo[3,4-d]pyrimidine or 4-(6-chloro-1-R-1H-pyrazolo[3,4-d]pyrimidine-4-yl)amino-phenylthiol were connected to pleuromutilin. A diverse array of substituents was introduced at the N-1 position of the pyrazole ring. The in vitro antibacterial activities of these semisynthetic derivatives were evaluated against two standard strains, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Staphylococcus aureus (S. aureus), ATCC 29213 and two clinical S. aureus strains (144, AD3) using the broth dilution method. Compounds 12c, 19c and 22c (MIC = 0.25 μg/mL) manifested good in vitro antibacterial ability against MRSA which was similar to that of tiamulin (MIC = 0.5 μg/mL). Among them, compound 22c killed MRSA in a time-dependent manner and performed faster bactericidal kinetics than tiamulin in time–kill curves. In addition, compound 22c exhibited longer PAE than tiamulin, and showed no significant inhibition on the cell viability of RAW 264.7, Caco-2 and 16-HBE cells at high doses (≤8 μg/mL). The neutropenic murine thigh infection model study revealed that compound 22c displayed more effective in vivo bactericidal activity than tiamulin in reducing MRSA load. The molecular docking studies indicated that compound 22c was successfully localized inside the binding pocket of 50S ribosomal, and four hydrogen bonds played important roles in the binding of them.


Introduction
The development of bacterial resistance to available antibiotics has been growing at a high speed, becoming one of the greatest public health problems. Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most important pathogens, which was first identified in the 1960s [1,2]. MRSA can cause a variety of diseases, ranging from skin infections to life-threatening invasive infections, such as pneumonia, endocarditis and sepsis [3]. MRSA infections are associated with high mortality [4]. There were about 120,000 bloodstream infections caused by MRSA in the USA in 2017, and nearly 20,000 of those affected lost their lives [5]. MRSA has been reported to be resistant to many clinical antibiotics, including linezolid, vancomycin, penicillin and all commonly prescribed betalactam antibiotics [6]. With the prevalence of MRSA, new antimicrobial agents are urgently needed to address its increasingly serious drug resistance.
Pleuromutilin (1, Figure 1), containing a tricyclic core of five-, six-and eight-membered rings, was first isolated from two basidiomycete species Pleurotus mutilus and Pleurotus passeckerianus in 1951 [7]. Pleuromutilin has been proven to be effective against Grampositive pathogens [7]. It has been identified that pleuromutilin inhibits bacterial protein Pleurotus passeckerianus in 1951 [7]. Pleuromutilin has been proven to be effective against Gram-positive pathogens [7]. It has been identified that pleuromutilin inhibits bacterial protein synthesis through its interaction with the 50S ribosomes [8,9]. This distinctive antibacterial mechanism endowed pleuromutilin with great potential to deal with drug-resistant bacterial infections, and encouraged researchers to develop novel pleuromutilin derivatives as effective antibacterial agents [10]. It was found that the modification of the C-14 acyloxy side chain significantly affected the antibacterial activity of pleuromutilin [11]. Based on this modification strategy, four of them have reached the market: tiamulin (2, Figure 1), valnemulin (3, Figure 1), retapamulin (4, Figure 1) and lefamulin (5, Figure  1) [12,13]. Tiamulin and valnemulin have been used to treat economically important infections in swine and poultry, which were approved in 1979 and 1999, respectively [14,15]. Success in veterinary antibiotics has encouraged researchers to focus on the development of new pleuromutilin antibiotics for human use. Retapamulin is the first pleuromutilin antibiotic for human skin infections caused by S. aureus [16]. In 2019, lefamulin was approved as an oral and intravenous pleuromutilin antibiotic for the treatment of community-acquired bacterial pneumonia in humans [17].  (4), lefamulin (5) ibrutinib (6), formycin A (7) and pyrazolo [3,4-d]pyrimidine (8).
Our lab is devoted to the development of pleuromutilin derivatives containing nitrogenous bases in the C14 side chain. Previous work has led to quite a few target products with powerful anti-MRSA activity. Fused N-heterocycles are important heterocyclic compounds with a wide spectrum of bioactivities, such as anti-cancer (ibrutinib, 6, Figure 1) and anti-bacterial (formycin A, 7, Figure 1) [18][19][20][21]. Among them, pyrazolo [3,4-d]pyrimidine (8, Figure 1) derivatives possess antibacterial pharmacological activity [22,23]. These motivated us to develop pleuromutilin derivatives contenting pyrazolo [3,4-d]pyrimidine moiety (Scheme 1). Additionally, the introduction of 4-aminothiophenol has been reported to influence the antimicrobial activity of pleuromutilin [24]. Thus, another synthetic strategy is              As illustrated in Tables 1 and 2, the antimicrobial activity of most compounds was absent. Compounds 12c, 19c and 22c (MIC = 0.25 μg/mL) exhibited better anti-bacterial effects than tiamulin (MIC = 0.5 μg/mL). Meanwhile, the preliminary structure-activity relationships (SARs) were also studied. Two schemes were designed to investigate the effect of the thioether bonds at the C-14 side chain on the antimicrobial activity of the pleuromutilin derivative. The oxygen atom at the C-22 position of the pleuromutilin was replaced by the nitrogen atom to furnish compounds 12b~28b in Scheme 1. MIC values of most compounds in Scheme 1 were not found (higher than 64 μg/mL), while compound 12b exhibited a better antibacterial effect against MRSA (MIC = 8 μg/mL). In Scheme 2, the oxygen atom at the C-22 position of the pleuromutilin was replaced by the sulfur atom, namely the sulphydryl at 1 position on 4-aminothiophenol, resulting in the formation of the thioether bond. As outlined in Table 2 As illustrated in Tables 1 and 2, the antimicrobial activity of most compounds was absent. Compounds 12c, 19c and 22c (MIC = 0.25 µg/mL) exhibited better anti-bacterial effects than tiamulin (MIC = 0.5 µg/mL). Meanwhile, the preliminary structure-activity relationships (SARs) were also studied. Two schemes were designed to investigate the effect of the thioether bonds at the C-14 side chain on the antimicrobial activity of the pleuromutilin derivative. The oxygen atom at the C-22 position of the pleuromutilin was replaced by the nitrogen atom to furnish compounds 12b~28b in Scheme 1. MIC values of most compounds in Scheme 1 were not found (higher than 64 µg/mL), while compound 12b exhibited a better antibacterial effect against MRSA (MIC = 8 µg/mL). In Scheme 2, the oxygen atom at the C-22 position of the pleuromutilin was replaced by the sulfur atom, namely the sulphydryl at 1 position on 4-aminothiophenol, resulting in the formation of the thioether bond. As outlined in Table 2, the MIC of compound 32 against MRSA was 0.0015. Compared with Scheme 1, compounds 12c, 19c and 22c displayed enhanced antibacterial activity with the MIC values of 0.25 µg/mL against MRSA. The introduction of 4-aminothiophenol caused the formation of a sulfide structure at the C22 position, the antibacterial activity of the derivatives was significantly promoted. This is consistent with our previous findings that the thioether bond has a positive influence on the antimicrobial effect of pleuromutilin derivatives [31][32][33][34][35]. Furthermore, the substituents at the N1 position of the pyrazolo [3,4-d]pyrimidine ring contained aromatic rings with different substituents. A variety of electron-donating (methyl, ethyl) and electron-withdrawing (fluoro, chlorine, etc.) groups were introduced on the aromatic rings. The meta-methyl-substituted compound 12c (0.25 µg/mL) showed superior antibacterial activities than compounds 13c~17c against MRSA. Compounds 18c, 20c and 21c with ortho-fluoro-substituted benzene derivatives exhibited much less potency compared to compounds 19c and 22c in this series. Furthermore, compound 23c, bearing a trifluoromethoxy group in the 4-position, showed activity comparable to tiamulin (MIC was 0.5 µg/mL) against MRSA. Generally, the ortho-position appeared to have less positive influence on the antibacterial activity of these pleuromutilin derivatives bearing a phenyl group.
This may be due to the pyrazolo [3,4-d] pyrimidine ring and their substituent groups forming a rigid planar structure. There were a large number of hydrogen bond donors, such as N, O and F. This may prevent the extension of the C14 side chain of pleuromutilin to PTC of 50S ribosomes, because it does not have a flexible conformation. At the same time, a large number of hydrogen bond donors in their structure must be disturbed by environmental substances when the compounds produce effects, violating the Lipinski rules. Thus, the introduction of this pyrimidine ring at the C14 side chain of pleuromutilin may not be a good modification strategy.
MBC refers to the minimum drug concentration required to kill 99.9% of the test microorganisms [33]. The MBC/MIC ratios of compounds 12c, 19c and 22c against MRSA were ≤2. According to previous studies, an antimicrobial drug can be considered bactericidal when the MBC/MIC is ≤4 [36]. Thus, the antimicrobial activity of compounds 12c, 19c and 22c was investigated for in-depth study.
Time-kill kinetic assays were performed, which were used to investigate the in vitro bactericidal kinetic effect of compounds 12c, 19c, 22c and tiamulin. The results are presented in Figure 2.
PAE has been considered as pharmacodynamic assistance which provides a reference for the rational formulation of dosing regimen [38]. PAE refers to the temporary suppression of bacterial growth following transient antibiotic treatment [39]. PAE assays were conducted for compounds 12c and 22c. The results were shown in Table 3, and the bacterial growth kinetics curves were exhibited in Figure 3. The test compounds displayed a significant inhibitory effect against MRSA at 1 × MIC. After 24 h incubation, compound 19c and tiamulin showed bactericidal effects, killing 99.9% of MRSA (−3.57 log10 CFU/mL and −3.78 log10 CFU/mL reduction, respectively) at 2 × MIC. After incubation for 24 h, compounds 12c and 22c displayed bactericidal ef-  fects, killing 99.99% of MRSA (−4.51 log10 CFU/mL and −4.47 log10 CFU/mL reduction, respectively) at 2 × MIC. Compared with tiamulin, compounds 12c and 22c manifested faster bactericidal kinetics against MRSA. Compounds 12c, 19c and 22c induced MRSA killing significantly at 4 × MIC (−5.76 log10 CFU/mL, −5.16 log10 CFU/mL and −5.79 log10 CFU/mL reduction, respectively). However, after the test compounds reached a certain concentration, the bactericidal effect did not significantly increase. The results indicated that compounds 12c, 19c, 22c and tiamulin are time-dependent drugs rather than concentrationdependent drugs. In clinical practice, multiple or continuous intravenous time-dependent antimicrobial agents can achieve better therapeutic results [37]. PAE has been considered as pharmacodynamic assistance which provides a reference for the rational formulation of dosing regimen [38]. PAE refers to the temporary suppression of bacterial growth following transient antibiotic treatment [39]. PAE assays were conducted for compounds 12c and 22c. The results were shown in Table 3, and the bacterial growth kinetics curves were exhibited in Figure 3.    PAE is calculated from growth curves on the difference in time required for the number (1 log 10 CFU/mL) of drug-exposed and unexposed microbes [40]. Following exposure to compounds 12c and 22c at the concentration of 2 × MIC for 1 h, the corresponding PAE values were 0. 37  Results obtained from the PAE assays showed that compounds 12c and 22c had been speculated to possess longer administration intervals than tiamulin. There is still a certain antibacterial effect as the drug concentration drops below the MIC. In clinical medication, pharmacodynamic parameters such as PAE, MIC and MBC can be used as references to evaluate the efficacy of antimicrobial agents. PAE provides a theoretical basis for the adjustment of the time interval to reduce adverse drug effects [41].

Cytotoxicity Assay
The presence of compounds can influence cellular basic physiological processes, suppress proliferation and even reduce cell survival, etc. In the community, MRSA generally causes respiratory infection [42]. Therefore, cytotoxicity was evaluated to explore the effect of compounds 12c and 22c on the viability of the respiratory tract cells 16-HBE (human bronchial epithelial cell line). RAW 264.7 (mouse peritoneal macrophage cell line) and Caco-2 (human epithelial colorectal adenocarcinoma cell line) were also used to evaluate the cytotoxicity of compounds 12c and 22c by MTT assay.
As shown in Figure 4, compound 22c displayed slight inhibition on the viability of RAW 264.7 cells at the concentration of 8 µg/mL. Compounds 12c and 22c did not affect the viability of Caco-2 and 16-HBE cells at the concentrations of 1~8 µg/mL. The study indicated that compounds 12c and 22c possessed a safety profile towards RAW 264.7 cells, Caco-2 and 16-HBE cells at higher doses.

Neutropenic Murine Thigh Infection Model
Compounds 12c and 22c possessed superior in vitro activity against MRSA and had been identified to be non-cytotoxic on RAW 264.7, Caco-2 and 16-HBE cells at high doses. Therefore, the in vivo efficacy of compounds 12c and 22c were assessed using the murine neutropenic thigh infection model. Mice groups treated with saline and tiamulin were chosen as the negative and positive controls. The results are shown in Figure 5.
Compared to the growth control group, tiamulin could reduce MRSA load (−0.56 log 10 CFU/mL) against MRSA in thigh muscle (p < 0.0001, n = 6/group). Compounds 12c and 22c at the same dose could reduce the MRSA load (−0.79 log 10 CFU/mL and −0.93 log 10 CFU/mL, respectively) in thighs compared with the blank control group (p < 0.0001, n = 6/group, both). Compounds 12c and 22c displayed more effective bactericidal activity than tiamulin in reducing MRSA load in thigh-infected mice (p = 0.0009 and 0.0001, respectively). This indicates preliminarily that compound 22c had a better in vivo anti-MRSA effect than compound 12c and tiamulin in the neutropenic murine thigh infection model, which could be used as a drug candidate against MRSA.

Molecular Docking Study
To predict the binding conformations of compound 22c to 50S ribosomes, molecular docking experiments were conducted [43]. The crystal structure (PDB: 1XBP) was obtained from the RCSB Protein Data Bank [44]. The validation of the docking scheme was performed by evaluating the quantitatively root mean square deviation (RMSD) of atom positions between the docking pose (the test compound to 1XBP) and X-ray crystallographic conformation (tiamulin to 1XBP). The quality of protein-ligand interactions can be expressed to some extent by the ligand efficiency (LE), the average binding free energy per non-hydrogen (or heavy) atom of the ligand [44]. The RMSD of compound 22c was 0.921 Å. Compounds 22c and tiamulin showed similar binding modes with 50S ribosomes, which were presented in Figure 6a.
The binding free energy of compound 22c with 50S ribosome was −7.11 kcal/mol. As shown in Figure 6b, four strong hydrogen bonds were found through the interaction of compound 22c and the 50S ribosome, namely 3-O and G2044 (distance: 2.2 Å, 2.2 Å), 11-O and G2484 (distance: 2.7 Å) and 21-O and G2482 (distance: 2.4 Å). The result predicted the binding mode of compound 22c to the 50S ribosome to indicate that they might have a good affinity.
As shown in Figure 4, compound 22c displayed slight inhibition on t RAW 264.7 cells at the concentration of 8 μg/mL. Compounds 12c and 22c the viability of Caco-2 and 16-HBE cells at the concentrations of 1~8 μg/m indicated that compounds 12c and 22c possessed a safety profile towards RA Caco-2 and 16-HBE cells at higher doses.

Neutropenic Murine Thigh Infection Model
Compounds 12c and 22c possessed superior in vitro activity against MRSA a been identified to be non-cytotoxic on RAW 264.7, Caco-2 and 16-HBE cells at hig Therefore, the in vivo efficacy of compounds 12c and 22c were assessed using the neutropenic thigh infection model. Mice groups treated with saline and tiamul chosen as the negative and positive controls. The results are shown in Figure 5.

Materials
Pleuromutilin (>90% pure) was purchased from Great Enjoyhood Biochemical Co., Ltd., (Daying, China). Urea, sodium methanolatea and hydrochloric acid were purchased from Titan Technology Co., Ltd., (Shanghai, China). Diethyl malonate and hydrazine derivatives were purchased from Bide Technology Co., Ltd., (Shanghai, China). The other analytical-grade solvents were purchased from Guangzhou General Reagent Factory (Guangzhou, China). Chromatographic purification was carried out on silica gel columns (200-300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd., Shandong, China). 1 H NMR and 13 C NMR spectra were measured on a Bruker AV-600 spectrometer in chloroform-d or DMSO-d 6 . Tetramethylsilane was used as the internal standard. Chemical shift values (δ) were indicated as ppm. High-resolution mass spectrometry was performed using a Thermo Scientific Q Exactive Focus Orbitrap LC-MS/MS with an electrospray ionization (ESI) source.

4,6-Dichloro-1-R-1H-pyrazolo[3,4-d]pyrimidine (12a~28a)
The raw material for synthesizing barbituric acid (compound 10) was urea (compound 9). Urea powder (6 g, 100 mmol) was dissolved in EtOH (50 mL), and then diethyl malonate (16 g, 120 mmol) was added to the solution. The reaction mixture was incubated with sodium methanolate (6.48 g, 120 mmol) for 48 h at 65 • C. The solution was acidified to pH 1~2, cooled down, crystallized and recrystallized to obtain pure barbituric acid. The Vilsmeier-Haack reaction of barbituric acid provided the product 2,4,6-trichloropyrimidine-5-carbaldehyde (compound 11). Phosphorus oxychloride (17.9 g, 117 mmol) was added into a 3-neck boiling flask at −10 • C, then N,N-dimethylformamide (15 mL) was dropped slowly into Phosphorus oxychloride, and then stirred for 1 h. Afterwards, Barbituric acid (5 g, 39 mmol) was added to the mixture and stirred for 48 h at 100 • C. After the reaction finished, the solution was slowly added into the ice water, a large number of solid precipitated out and then filtered, and the yield of compound 11 was 92%. The pyrazole ring was closed by treatment with hydrazine derivatives. Hydrazine derivatives (5.64 mmol) and compound 11 (1 g, 4.7 mmol) were dissolved in EtOH (10 mL) and then stirred for 2 h at −20 • C under alkaline conditions. After the reaction was completed, a large amount of solid was precipitated from the reaction solution. The crude products were purified using EtOH to obtain compounds 12a~28a. Yield: 83%~98%. (31) 4-methylbenzene-1-sulfonyl chloride (5.6 g, 29.2 mmol) and pleuromutilin (10 g, 26.5 mmol) were dissolved in acetonitrile (50 mL), then sodium hydroxide granules (3 g, 52.84 mmol) were dissolved in water (15 mL) and dropped slowly into the above mixture solution and stirred in an ice bath for 3 h. The mixture was then vacuum-evaporated, extracted with 30 mL of dichloromethane and washed with water (30 mL). The organic layer was dried over anhydrous Na 2 SO 4 and filtered. Afterwards, isopropanol (50 mL) was added, and the mixture was heated at 70 • C until the solid was completely dissolved. Stewing the solution at room temperature for 1-2 h, the white solid precipitated and the white solid was collected (compound 29), yield: 95%.

22-Amino-deoxypleuromutilin
Compound 29 (1 g, 1.88 mmol) and sodium azide (0.37 g, 5.65 mmol) were added to 10 mL of acetone and 5 mL of water, respectively. The two solutions were mixed under continuous stirring and heated at 80 • C for 4 h. The mixture was then vacuumevaporated, extracted with 30 mL of dichloromethane and washed with water (30 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain the product compound 30, yield: 93%. Compound 30 (10 g, 25.67 mmol) and triphenylphosphine (7.14 g, 28.24 mmol) were dissolved in THF (80 mL) and H 2 O (20 mL) solution. The mixture was maintained in an ice bath for 2 h, then washed with dichloromethane (30 mL) and water (50 mL) 3 times. The organic phase was dried over anhydrous Na 2 SO 4 and evaporated in vacuum. The crude product was purified by column chromatography (dichloromethane: methanol = 200:1) using silica gel to obtain compound 31.

22-[(6-Chloro-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4-yl)amino]-22-
After obtaining the MIC results, the 96-well plates were incubated at 37 • C for 24 h. Overall, 25 µL of the bacterial solution from the wells with no obvious bacterial growth was inoculated on MH agar plates [45]. Then, the inoculated MH agar plates were further cultured at 37 • C for 24 h. MBC was defined as the minimum concentration at which bacterial growth was not observed (99.9% of the bacteria were killed).

Constant Concentration Time-Kill Curves
Time-kill curve assays were established with the MIC value to investigate the antibacterial effect of constant drug concentration on MRSA. The antibiotic concentrations were equal to 1 × MIC, 2 × MIC, 4 × MIC, 8 × MIC, 16 × MIC and 32 × MIC as described. MRSA ATCC 43300 was incubated in MH broth at 37 • C for 4.5 h and diluted to 1 × 10 6 CFU/mL in MH broth. The saline control group was subjected to the same schedule but 0.9% saline water was added instead of the test compound. All the samples were incubated in an oscillating thermostatic at 37 • C, and then 100 µL of the mixture was extracted to 900 µL of sterile saline (0.9%) at 0, 3, 6, 9, 12 and 24 h, respectively. Samples were serially diluted 10-fold in sterile saline (0.9%) and inoculated onto agar plates. The colonies were counted after incubation at 37 • C for 24 h. Three independent experiments were performed according to our previous work [34]. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time, and the change in bacterial concentration was determined.

Determination of the Post-Antibiotic Effect (PAE)
PAE of compounds 12c and 22c on MRSA ATCC 43300 was determined using MH broth according to our previous work [46]. The final concentration of MRSA was 1 × 10 6 CFU/mL by dilution with MH broth. Compounds 12c, 22c and tiamulin were supplemented in the suspension at a final concentration of 2 × MIC and 4 × MIC, respectively. The negative control group contained untreated MRSA bacterial cells. The test tubes were incubated with a 37 • C constant temperature vibration incubator for 1 and 2 h. After incubation, the test compound was removed from the sample by diluting 1000-fold with the preheated MH broth. Next, 100 µL of suspension from each culture was 10-fold diluted in sterile saline and inoculated on MH agar plates at 0, 2, 4, 6 and 8 h after inoculation. The number of colonies was calculated after incubation at 37 • C for 24 h. The experiments were performed in triplicate.

Cytotoxicity Assay
Cytotoxicity of the test compounds was assessed using the conventional MTT method [46]. RAW 264.7 murine macrophage cells, Caco-2 cells and 16-HBE were used in this experiment. The cells were seeded into 96-well plates at a density of 1.0 × 10 5 cells per well. After 4 h at 37 • C, the cells were treated with compounds 12c and 22c at various concentrations and incubated at 37 • C for 16 h. Next, MTT (0.5 mg/mL in PBS, 100 µL/well) was added sequentially to each well in a humidified atmosphere of 95% air and 5% CO 2 incubator and the incubation continued for an additional four hours. After incubation, the medium was peeled off and DMSO (150 µL/well) was added to dissolve the cells, and then the incubation was continued for 30 min. At the end of this period, the absorbance was measured at 490 nm by a microplate spectrophotometer (BIO-TEK Instruments, Winooski, VT, USA).

Neutropenic Murine Thigh Infection Model
Model experiments of neutropenic mouse thigh infection were manipulated as described in the literature [32]. Female, six-week-old, specific-pathogen-free mice weighing approximately 23~26 g were used throughout the study. Mice were rendered neutropenic by injection of cyclophosphamide (Mead Johnson Pharmaceuticals, Evansville, IN) on day 4 (150 mg/kg) and day 1 (100 mg/kg) before the experiment. The neutrophil numbers in mice blood should be <0.1 × 10 9 /L [34]. In total, 0.1 mL of MH broth of MRSA (10 7 CFU/mL) was inoculated into each thigh of the neutropenic mice. Mice were divided into 4 groups (6 mice per group), including control (sterile saline), compounds 12c, 22c and tiamulin groups. After 3 h post-infection, the corresponding drugs were injected into each mouse's thigh. Mice were euthanized following intravenous injection for 24 h. Thigh tissue of each mouse was removed, collected, weighed and homogenized in 3 mL of ice sterile saline. Six ten-fold serial dilutions were performed and 25 µL of the bacterial solution from each tube was plated on MH agar plates. The resulting colonies were counted after 24 h incubation at 37 • C. The protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee of the South China Agricultural University.

Molecular Modeling
Docking studies were carried out based on the binding mode of the Staphylococcus aureus 50S ribosome with tiamulin (PDB ID code: 1XBP). The binding pattern of compound 22c to S. aureus 50S ribosome was investigated. All residues within 40 Å around tiamulin in 1XBP were built as a peptidyl transferase centre (PTC) model. The model was then refined using a standard energy minimization protocol. The test compound was prepared by Avogadro 1.1.1, with a 5000-step Steepest Descent as well as a 1000-step Conjugate Gradients geometry optimization using MMFF94 force field. Docking experiments were performed using AutoDock, Vina and Pymol [47].

Conclusions
Two series of pleuromutilin derivatives containing 6-chloro-4-amino-1-R-1H-pyrazolo [3,4d]pyrimidine structures were synthesized and evaluated as inhibitors against MRSA. The present study revealed the synthesis procedure, antibacterial activity and cytotoxicity of the designed compounds. Compound 22c in Scheme 2 displayed better antibacterial activity against MRSA than tiamulin. The resulting time-kill curve experiments indicated that compound 22c was time-dependent rather than dose-dependent and manifested a more rapid bactericidal kinetic effect than tiamulin. Compound 22c performed longer PAE than tiamulin, indicating a longer administration interval than tiamulin. The results of the cytotoxicity assay revealed that compound 22c exhibited no significant inhibitory effect on RAW 264.7 cells, Caco-2 cells and 16-HBE cells at high doses. Meanwhile, compound 22c exhibited more potent in vivo bactericidal effects than tiamulin in the neutropenic murine thigh infection model studies. Moreover, the molecular docking studies indicated that four hydrogen bonds played important roles in the binding of compound 22c to 50S ribosomes. This study indicated that compound 22c was worthy of further development as a potential drug against MRSA infection.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28093975/s1. Figure S1-S102: The characterization spectrum of synthesized compounds;  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. The study did not involve humans. This experiment protocols were reviewed and approved by the Committee on the Ethics of Animals of South China Agricultural University (Approval number: 2021C061).
Data Availability Statement: Data available on request due to restrictions privacy. The data presented in this study are available on request from the corresponding author.